After DNA is transcribed and translated into a protein, further post-translational processing involves the attachment of sugar residues, a process known as glycosylation. Different organisms produce different glycosylation enzymes (glycosyltransferases and glycosidases), and have different substrates (nucleotide sugars) available, so that the glycosylation patterns as well as composition of the individual oligosaccharides, even of the same protein, will be different depending on the host system in which the particular protein is being expressed. Bacteria typically do not glycosylate proteins, and if so only in a very unspecific manner. Lower eukaryotes such as filamentous fungi and yeast add primarily mannose and mannosylphosphate sugars. The resulting glycan is known as a “poly-mannose” type glycan or a mannan.
By contrast, in higher eukaryotes such as humans, plant cells and insect cells, the nascent oligosaccharide side chain may be trimmed to remove several mannose residues and elongated with additional sugar residues that typically are not found in the N-glycans of lower eukaryotes such as filamentous fungi and yeast. This synthesis begins with a set of sequential reactions in the course of which sugar residues are added and removed while the protein moves along the secretory pathway in the host organism. However, enzymes which reside in the Golgi apparatus of the host organism or cell differ in their specificities and thus determine the resulting glycosylation patterns of secreted proteins.
Thus, the resulting glycosylation pattern of proteins expressed in eukaryotic host cells such as yeast, plants or insects, differs substantially from the glycosylation pattern of proteins expressed in humans and other mammals.
The early steps of the mammalian protein glycosylation can be divided into at least four different phases:    (i) lipid-linked Glc3Man9GlcNAc2 oligosaccharides are assembled by a sequential set of reactions at the membrane of the endoplasmic reticulum (ER) and    (ii) transfer of this oligosaccharide from the lipid anchor dolichol pyrophosphate onto de novo synthesized protein. The site of the specific transfer is defined by an asparagine (Asn) residue in the sequence Asn-Xaa-Ser/Thr where Xaa can be any amino acid except proline and aspartic acid.    (iii) Further processing by glucosidases and mannosidases occurs in the ER before the nascent glycoprotein is transferred to the cis-Golgi apparatus, where additional mannose residues are removed by Golgi specific α1,2-mannosidases.    (iv) Processing continues as the protein proceeds through the Golgi apparatus. In the median-Golgi, a number of modifying enzymes, including N-acetylglucosaminyltransferases (GnTI, GnTII, GnTIII, GnTIV and GnTV), mannosidase II and fucosyltransferases, add and remove specific sugar residues. Finally, in the trans-Golgi, galactosyltranferases (GalT) and sialyltransferases (ST) produce a glycoprotein structure that is released from the Golgi. It is this structure, characterized by bi-, tri- and tetra-antennary structures, containing galactose, fucose, N-acetylglucosamine and a high degree of terminal sialic acid that gives glycoproteins their mammalian characteristics.
In nearly all eukaryotes, glycoproteins are derived from a common lipid-linked oligosaccharide precursor Glc3Man9GlcNAc2-dolichol-pyrophosphate. Within the endoplasmic reticulum, synthesis and processing of dolichol pyrophosphate bound oligosaccharides are identical between all known eukaryotes.
However, further processing of the core oligosaccharide by fungal, plant or insect cells once it has been transferred to a peptide leaving the ER and entering the Golgi, differs significantly from humans as it moves along the secretory pathway and involves in the Golgi apparatus the addition of several organism-specific sugars.
A significant fraction of proteins isolated from humans or other animals are glycosylated. Among proteins used therapeutically, about 70% are glycosylated. If a therapeutic protein is produced in an organism host such as yeast or fungus, however, and is glycosylated utilizing the endogenous pathway, its therapeutic efficiency is typically greatly reduced. Such glycoproteins are typically immunogenic in humans and show a reduced half-life in vivo after administration. Specific receptors in humans and animals can recognize terminal mannose residues and promote the rapid clearance of the protein from the bloodstream. Additional adverse effects may include changes in protein folding, solubility, susceptibility to proteases, trafficking, transport, compartmentalization, secretion, biological activity, recognition by other proteins or factors, antigenicity, or allergenicity. Accordingly, it has been necessary to produce therapeutic glycoproteins in animal host systems, so that the pattern of glycosylation is identical or at least similar to that, in humans or in the intended recipient species. In most cases a mammalian host system, such as mammalian cell culture, is used.
In order to produce therapeutic proteins that have appropriate glycoforms and have satisfactory therapeutic effects, animal or plant-based expression systems have been used. The available systems include:                Chinese Hamster Ovary cells (CHO), mouse fibroblast cells and mouse myeloma cells;        transgenic animals such as goats, sheep, mice and others;        yeast (such as S. pombe, S. cerevisiae, P. pastoris), bacteria (such as E. coli), fungi (such as A. nidulans, T ressei);—plants (such as A. thaliana, N. tabacum, M. sativa etc.);        insect cells (such as S. frugiperda Sf9, Sf21, Trichoplusia ni, etc. in combination with recombinant baculoviinses such as A. californica multiple nuclear polyhedrosis virus which infects lepidopteran cells).        
Recombinant human proteins expressed in the above-mentioned host systems may still include non-human glycoforms. In particular, fraction of the N-glycans may lack terminal sialic acid, typically found in human glycoproteins. Substantial efforts have been directed to develop processes to obtain glycoproteins that are as close as possible in structure to the human forms, or have other therapeutic advantages such as having specific glycoforms that may be especially useful, for example in the targeting of therapeutic proteins. For example, the addition of one or more sialic acid residues to a glycan side chain may increase the lifetime of a therapeutic glycoprotein in vivo after administration. Accordingly, the mammalian host cells may be genetically engineered to increase the extent of terminal sialic acid in glycoproteins expressed in the cells. Alternatively sialic acid may be conjugated to the protein of interest in vitro prior to administration using a sialyltransferase and an appropriate substrate. In addition, changes in growth medium composition or the expression of enzymes involved in human glycosylation have been employed to produce glycoproteins more closely resembling to the human forms. Alternatively cultured human cells may be used.
However, all of the existing systems have significant drawbacks. Only certain therapeutic proteins are suitable for expression in animal or plant systems (e.g. those lacking in any cytotoxic effect or other effect adverse to growth).
Animal and plant cell culture systems may be very slow, frequently requiring up to a week of growth under carefully controlled conditions to produce any useful quantity of the protein of interest. Protein yields nonetheless compare unfavorably with those from microbial fermentation processes. In addition, animal cell culture systems typically require complex and expensive nutrients and cofactors, such as bovine fetal serum. Furthermore growth may be limited by programmed cell death (apoptosis).
Moreover, animal cells (particularly mammalian cells) are highly susceptible to viral infection or contamination. In some cases, virus or other infectious agent may just compromise the growth of the culture, while in other cases; this agent may be a human pathogen rendering the therapeutic protein product unfit for its intended use. Furthermore many cell culture processes require the use of complex, temperature-sensitive, animal-derived growth media components, which may carry pathogens such as bovine spongiform encephalopathy (BSE) prions. Such pathogens are difficult to detect and/or difficult to remove or sterilize without compromising the growth medium. In any case, use of animal cells to produce therapeutic proteins necessitates costly quality controls to assure product safety.
Transgenic animals may also be used for manufacturing high-volume of therapeutic proteins such as human serum albumin, tissue plasminogen activator, monoclonal antibodies, hemoglobin, collagen, fibrinogen and others. While transgenic goats and other transgenic animals (mice, sheep, cows, etc.) can be genetically engineered to produce therapeutic proteins at high concentrations in the milk, the process is costly since every batch has to undergo rigorous quality control. Animals may host a variety of animal or human pathogens, including bacteria, viruses, fungi, and prions. In the case of scrapies and bovine spongiform encephalopathy, testing can take about a year to rule out infection. The production of therapeutic compounds is thus preferably carried out in a well-controlled sterile environment, e.g. under Good Manufacturing Practice (GMP) conditions. However, it is not generally feasible to maintain animals in such environments. Moreover, whereas cells grown in a fermenter are derived from one well characterized Master Cell Bank (MCB), transgenic animal technology relies on different animals and thus is inherently non-uniform. Furthermore external factors such as different food uptake, disease and lack of homogeneity within a herd, may effect glycosylation patterns of the final product. It is known in humans, for example, that different dietary habits result in differing glycosylation patterns.
Transgenic plants have been developed as a potential source to obtain proteins of therapeutic value. However, high level expression of proteins in plants suffers from gene silencing, a mechanism by which the genes for highly expressed proteins are down-regulated in subsequent plant generations. In addition, plants add β(1,2)-linked xylose and/or α(1,3)-linked fucose to protein N-glycans, resulting in glycoproteins that differ in structure from animals and are immunogenic in mammals. Furthermore, it is generally not practical to grow plants in a sterile or GMP environment, and the recovery of proteins from plant tissues is more costly than the recovery from fermented microorganisms.
Transgenic yeast or fungi systems also present the drawback of expressing mannosyltransferase genes which adds a mannose to the glycan structure and leads to hypermannosylated proteins.
In conclusion, all different systems described here above present important drawbacks in terms of immunogenicity of the glycosylated proteins produced.
An object of the invention is therefore to provide an alternative and effective system for producing recombinant glycoproteins having a glycosylation pattern suitable for therapeutic purpose.
The Applicant found surprisingly that microalgae such as Phaeodactylum tricornutum (P. tricornutum) are capable of producing polypeptides harbouring Man5GlcNAc2 to Man9GlcNA2 via their endogenous N-glycosylation machinery. Analysis of N-glycans from other representative microalgae also revealed the presence of high-mannose-type oligosaccharides on their proteins. Thus, microalgae present the advantage to allow the production of proteins with a certain glycosylation pattern without needing the suppression of genes responsible for the addition of immunogenic epitopes such as in plants or yeasts. This discovery was unexpected since microalgae were thought to produce proteins having a plant glycosylation pattern and, eventually, a yeast or fungi glycosylation pattern. This idea is well illustrated by the patent application PCT WO 2006/013572 which discloses the production of a glycosylated Hepatitis B S antigen (HBsAg) in red microalgae and suggests to “humanize” the glycosylation pattern of recombinants products synthesized in red microalgae (page 16, lines 4 to 8) by:                inactivating in said microalgae α-mannosidase I and N-acetylglucosaminyltransferase (page 16, line 29-33), which enzymes are implicated in yeast and in fungi in the addition of a mannose to the glycan structure leading to hypermannosylated proteins); and        inactivating α(1,3)-fucosyltransferase and β(1,2)xylosyltransferase (page 17, lines 16-22 and lines 1-5), which enzymes are implicated in plants in the addition of β(1,2)-linked xylose and α(1,3)-linked fucose to protein N-glycans.        
Microalgae present also the advantage of being cultivated in confined photobioreactors, therefore overcoming the problem of gene dissemination into the environment and the problem of virus transmission to animals. In addition, microalgae culture system is very fast, provides an excellent yield in biomass and only requires sea water or fresh water, nutritive elements, carbon and light.